Methods for detecting immune cell DNA and monitoring immune system

Abstract

The disclosure herein provides methods and compositions for detecting or monitoring immune cell populations in biological samples. The methods and compositions disclosed herein are particularly useful for detecting or monitoring immune cell populations in patients suffering from a disease or undergoing treatment of a disease resulting in depletion of immune cells. In particular, the present disclosure provides method for using multiplex PCR combined with next-generation DNA sequencing to detect DNA containing recombined V(D)J gene segments which can be used to detect immune cells.

What is claimed is: 
     
       1. A method of preparing a non-naturally occurring composition of amplified DNA derived from a biological sample useful for detecting or monitoring immune cells in a subject, comprising:
 preparing a non-naturally occurring composition of amplified DNA by performing a multiplex amplification reaction on nucleic acids isolated from a biological sample of the subject to generate a set of amplicons, wherein each of the set of amplicons comprises recombined V(D)J gene segments at a gene locus of interest, wherein the multiplex amplification reaction is capable of amplifying at least about 70% of all possible V(D)J recombinations at the gene locus of interest, wherein the multiplex amplification reaction is performed with a first set of primers targeting V gene sequences targeted by one or more of the primers of SEQ ID NOs: 1-64 and a second set of primers targeting J gene sequences targeted by one or more of the primers of SEQ ID Nos: 65-76; and 
 analyzing the non-naturally occurring composition of amplified DNA by sequencing the set of amplicons, wherein sequences of the recombined V(D)J gene segments are indicative of presence of an immune cell in the biological sample. 
 
     
     
       2. The method of  claim 1 , wherein the multiplex amplification reaction is capable of amplifying at least about 90% of all possible V(D)J recombinations at the B cell receptor gene locus. 
     
     
       3. The method of  claim 1 , wherein the method comprises collecting and sequencing a plurality of biological samples from the subject longitudinally. 
     
     
       4. The method of  claim 1 , wherein the biological sample comprises a peripheral blood mononuclear cell (PBMCs) sample, a plasma sample, or a combination thereof. 
     
     
       5. The method of  claim 1 , wherein the nucleic acids isolated from the biological sample comprises cellular DNA obtained from PBMCs. 
     
     
       6. The method of  claim 1 , wherein the nucleic acids isolated from the biological sample comprises cell-free DNA (cfDNA). 
     
     
       7. The method of  claim 6 , wherein the amount of immune cells in the biological sample is less than 1.0%, less than 0.5%, or less than 0.1% of the PBMCs in the sample. 
     
     
       8. The method of  claim 1 , wherein the method is capable of detecting 5 or less V(D)J recombinations per milliliter of the biological sample. 
     
     
       9. The method of  claim 1 , wherein the method is capable of detecting a single V(D)J recombination per milliliter of the biological sample. 
     
     
       10. The method of  claim 1 , wherein the subject has been administered a cytotoxic treatment of a disease, wherein the cytotoxic treatment leads to depletion of the immune cells. 
     
     
       11. The method of  claim 10 , wherein the disease is a malignancy, and the cytotoxic treatment comprises a chemotherapy, a radiotherapy, or an immune cell targeted therapy; or wherein the disease is an autoimmune disease, and the cytotoxic treatment comprise an immunosuppressive therapy. 
     
     
       12. The method of  claim 1 , wherein the subject suffers from a disease or disorder that depletes immune cells, wherein the disease or disorder is a malignancy or an immunodeficiency disorder. 
     
     
       13. The method of  claim 1 , wherein the subject has been administered with a therapeutic composition comprising immune cells, and wherein the method further comprises analyzing sequences of the recombined V(D)J gene segments to determine the presence of the administered immune cells. 
     
     
       14. The method of  claim 1 , wherein the immune cell comprises a B cell, a transplanted B cell, an engineered B cell, a circulating bone marrow B cell, and/or a circulating tumor B cell. 
     
     
       15. The method of  claim 1 , further comprising analyzing sequences of the recombined V(D)J gene segments to determine a diversity of V(D)J recombination in the sample obtained from the subject and a control sample, wherein the diversity of V(D)J recombination is indicative of an immune receptor repertoire. 
     
     
       16. The method of  claim 15 , wherein the immune cell is a B cell, and the diversity of V(D)J recombination is indicative of the diversity of a B cell receptor (BCR) repertoire; or wherein the sample comprises a single isolated B cell or a clonal expansion thereof, and the diversity of V(D)J recombination is indicative of the diversity of the BCR repertoire of the single isolated B cell or the clonal expansion thereof.